There is a great need for developing genes that can be expressed in plants in order to provide transgenic insecticidal plants that effectively control various insects. Recently, considerable attention has been given to toxin complex (TC) genes, obtainable from a variety of organisms, including Photorhabdus, Xenorhabdus, Paenibacillus, Serratia, and Pseudomonas.
There has been substantial progress in the cloning of genes encoding insecticidal toxins from both Photorhabdus luminescens and Xenorhabdus nematophilus. Toxin-complex encoding genes from P. luminescens were examined first. See WO 98/08932. Parallel genes were more recently cloned from X. nematophilus. Morgan et al., Applied and Environmental Microbiology 2001, 67:20062-69; WO 95/00647 relates to the use of Xenorhabdus protein toxin to control insects, but it does not recognize orally active toxins. WO 98/08388 relates to orally administered pesticidal agents from Xenorhabdus. U.S. Pat. No. 6,048,838 relates to protein toxins/toxin complexes, having oral activity, obtainable from Xenorhabdus species and strains.
Four different toxin complexes (TCs)—Tca, Tcb, Tcc and Tcd—have been identified in Photorhabdus spp. Each of these toxin complexes resolves as either a single or dimeric species on a native agarose gel but resolution on a denaturing gel reveals that each complex consists of a range of species between 25-280 kDa. See WO 97/17432, WO 98/08932, and R. H. ffrench-Constant and Bowen, 57 Cell. Mol. Life Sci. 828-833 (2000).
Genomic libraries of P. luminescens were screened with DNA probes and with monoclonal and/or polyclonal antibodies raised against the toxins. Four tc loci were cloned: tca, tcb, tcc and tcd. The tca locus is a putative operon of three open reading frames (ORFs), tcaA, tcaB, and tcaC, transcribed from the same DNA strand, with a smaller terminal ORF (tcaZ) transcribed in the opposite direction. The tcc locus also is comprised of three ORFs putatively transcribed in the same direction (tccA, tccB, and tccC). The tcb locus is a single large ORF (tcbA), and the tcd locus is composed of two ORFs (tcdA and tcdB) ; tcbA and tcdA, each about 7.5 kb, encode large insect toxins. TcdB has some level of homology to TcaC. It was determined that many of these gene products were cleaved by proteases. For example, both TcbA and TcdA are cleaved into three fragments termed i, ii and iii (e.g. TcbAi, TcbAii and TcbAiii). Products of the tca and tcc ORFs are also cleaved. See WO 98/08932 and R. H. french-Constant and D. J. Bowen, Current Opinions in Microbiology, 1999, 12:284-288.
Bioassays of the Tca toxin complexes revealed them to be highly toxic to first instar tomato hornworms (Manduca sexta) when given orally (LD50 of 875 ng per square centimeter of artificial diet). R. H. french-Constant and Bowen 1999. Feeding was inhibited at Tca doses as low as 40 ng/cm2. Given the high predicted molecular weight of Tca, on a molar basis, P. luminescens toxins are highly active and relatively few molecules appear to be necessary to exert a toxic effect. R. H. ffrench-Constant and Bowen, Current Opinions in Microbiology, 1999, 12:284-288.
WO 99/42589 and U.S. Pat. No. 6,281,413 disclose TC ORFs from Photorhabdus luminescens. WO 00/30453 and WO 00/42855 disclose TC proteins from Xenorhabdus. 
While the exact molecular interactions of the TCs with each other, and their mechanism(s) of action, are not currently understood, it is known, for example, that the Tca toxin complex (comprised of TcaA, TcaB, and TcaC) of Photorhabdus is toxic to Manduca sexta. In addition, some TC proteins are known to have “stand alone” insecticidal activity, while other TC proteins are known to potentiate or enhance the activity of the stand-alone toxins. It is known that the TcdA protein is active, alone, against Manduca sexta, but that TcdB and TccC, together, can be used (in conjunction with TcdA) to greatly enhance the activity of TcdA. TcbA is the other main, stand-alone toxin from Photorhabdus. The activity of this toxin (TcbA) can also be greatly enhanced by TcdB- together with TccC-like proteins.
PhotorhabdusPhotorhabdusstrain W14TC proteinnomenclatureSome homology to:TcaAToxin CTccATcaBTccBTcaCTcdBTebAToxin BTccAToxin DTcdA N terminusTccBTcdA C terminusTccCTcdAToxin ATccA + TccBorTcaA + TcaBTcdBTcaC
TcaA, TcaB, TccA, and TccB are referred to as “small toxins”, as distinguished from the “large toxins” TcbA and TcdA. The pair TcaA+TcaB in Toxin C is analogous to the large toxin TcdA in Toxin A; and in the pair TccA+TccB in Toxin D is likewise analogous to the large toxin TcdA. N. R. Waterfield, et al., “The tc genes of Photorhabdus: a growing family,” TRENDS IN MICROBIOLOGY, Vol. 9, pp 185-191 (2001).
Some Photorhabdus TC proteins have some level of sequence homology with other Photorhabdus TC proteins. As indicated above, TccA has some level of homology with the N terminus of TcdA, and TccB has some level of homology with the C terminus of TcdA. Furthermore, TcdA is about 280 kDa, and TccA together with TccB are of about the same size, if combined, as that of TcdA. Though TccA and TccB are much less active on SCR than TcdA, TccA and TccB from Photorhabdus strain W14 are called “Toxin D.” “Toxin A” (TcdA), “Toxin B” (Tcb or TcbA), and “Toxin C” (TcaA and TcaB) are also indicated above.
Furthermore, TcaA has some level of homology with TccA and likewise with the N terminus of TcdA. Still further, TcaB has some level of homology with TccB and likewise with the N terminus of TcdA. TcdB has a significant level of similarity to TcaC.
Relatively recent cloning efforts in Xenorhabdus nematophilus also appear to have identified novel insecticidal toxin genes with homology to the P. luminescens tc loci. See, e.g., WO 98/08388 and Morgan et al, Applied and Environmental Microbiology 2001, 67:20062-69. In R. H. ffrench-Constant and D. J. Bowen Current Opinions in Microbiology, 1999, 12:284-288, cosmid clones were screened directly for oral toxicity to another lepidopteran, Pieris brassicae. One orally toxic cosmid clone was sequenced. Analysis of the sequence in that cosmid suggested that there are five different ORF's with similarity to Photorhabdus tc genes; orf2 and orf5 both have some level of sequence relatedness to both tcbA and tcdA, whereas orf1 is similar to tccB, orf3 is similar to tccC and orf4 is similar to tcaC. Importantly, a number of these predicted ORFs also share the putative cleavage site documented in P. luminescens, suggesting that active toxins may also be proteolytically processed.
Five typical TC proteins from Xenorhabdus have heretofore been identified: XptA1, XptA2, XptB1, XptC1, and XptD1. XptA1 and XptA2 were known to have stand-alone toxin activity. The XptA2 protein was known to have some degree of similarity to the TcdA protein. XptB1 and XptC1 are Xenorhabdus potentiators that were known to enhance the activity of either (or both) of the XptA toxins. XptD1 was known to have some level of homology with TccB, and XptC1 was known to have some level of similarity to TcaC. XptB1 has some level of similarity to TccC.
United States Patent Application 20040194164 of Scott B. Bintrim et al. on “Xenorhabdus TC proteins and genes for pest control” discloses a set of novel Xenorhabdus TC proteins and genes obtainable from the Xwi strain of Xenorhabdus nematophilus. It also provides an exochitinase obtainable from from the Xwi strain of Xenorhabdus nematophilus. 
TC proteins and genes have more recently been described from other insect-associated bacteria such as Serratia entomophila, an insect pathogen. Waterfield et al., TRENDS in Microbiology, Vol. 9, No. 4, April 2001.
TC proteins and lepidopteran-toxic Cry proteins have very recently been discovered in Paenibacillus. See U.S. Ser. No. 60/392,633 (Bintrim et al.), filed Jun. 28, 2002. Bacteria of the genus Paenibacillus are distinguishable from other bacteria by distinctive rRNA and phenotypic characteristics (C. Ash et al. (1993), “Molecular identification of rRNA group 3 bacilli (Ash, Farrow, Wallbanks and Collins) using a PCR probe test: Proposal for the creation of a new genus Paenibacillus,” Antonie Van Leeuwenhoek 64:253-260). Some species in this genus are known to be pathogenic to honeybees (Paenibacillus larvae) and to scarab beetle grubs (P. popilliae and P. lentimorbus.) P. larvae, P. popilliae, and P. lentimorbus are considered obligate insect pathogens involved with milky disease of scarab beetles (D. P. Stahly et al. (1992), “The genus Bacillus: insect pathogens,” p. 1697-1745, In A. Balows et al., ed., The Procaryotes, 2nd Ed., Vol. 2, Springer-Verlag, New York, N.Y.).
Although some Xenorhabdus TC proteins have been found to “correspond” (have a similar function and some level of sequence homology) to some of the Photorhabdus TC proteins, the “corresponding” proteins share only about 40% (approximately) sequence identity with each other. This is also true for the more recently discovered TC proteins from Paenibacillus (those proteins and that discovery are the subject of co-pending U.S. Ser. No. 60/392,633).
Some TC proteins have stand alone insecticidal activity, but it is known that this activity can be enhanced when such proteins are used in combination with other TC proteins. In this regard, three relevant classes of TC proteins have heretofore been identified: Class A proteins, Class B proteins, and Class C proteins. A protein belonging to one of these classes is referred to hereinafter as a “Protein A”, a “Protein B”, or a “Protein C”. Proteins are assigned to Class A, Class B, or Class C based on sequence similarity, as discussed in greater detail hereinafter. Class A proteins have stand alone insecticidal activity. Typical examples of Class A proteins are the “large toxins” TcdA, TcdA2, TcdA4, and TcBA from Photorhabdus luminescens, XptA1Xwi and XptA2Xwi from Xenorhabdus nematophilus, and SepA from Serratia entomophila. Class A proteins should also be understood to include the small toxin pairs that correspond to the large toxins, e.g. TcaA+TcaB in Toxin C and TccA+TccB in Toxin D. Class B and Class C proteins lack significant stand alone insecticidal activity, and have been referred to as potentiators. Typical Class B proteins are TcdB1, TcdB2, TcaC from Photorhabdus luminescens, XptC1Xwi Xenorhabdus nematophilus, PptB11529 from Paenibacilluss spp., and SepB from Serratia entomophila. Typical Class C proteins are TccC2 from Photorhabdus luminescens, XptB1Xwi from Xenorhabdus nematophilus, and XptC1Xb from Xenorhabdus bovienii. It has repeatedly been found that use of a Protein A in combination with a Protein B and a Protein C substantially enhances insecticidal activity over that obtained with the Protein A alone.
United States Patent Application Publication 2004/0208907 demonstrates that the activity of a Protein A can be potentiated by a Protein B and Protein C even if the Protein B and/or Protein C originates from an entirely distinct species from the one that produces the Protein A.
In light of concerns about insects developing resistance to a given pesticidal toxin, and in light of other concerns—some of which are discussed above, there is a continuing need for the discovery of new insecticidal toxins and other proteins that can be used to control insects.